The polypeptide hormone prolactin is synthesized by specialized cells, lactotrophs, in the anterior pituitary and is a major regulator of lactogenesis. The genes encoding several animal prolactins have been sequenced [human (Cooke et al., J. Biol. Chem., (1981) 256 (8):4007), bovine (Nilson et al., Nucleic Acids Research (1980) 8(7):1561), rat (Cooke et al., J. Biol. Chem., (1980) 255(13):6502)].
Studies using a rat pituitary tumor cell line that constitutively expresses prolactin and growth hormone have suggested that the synthesis of prolactin is regulated transcriptionally by a number of factors including cAMP, epidermal growth factor (EGF), phorbol esters, thyrotropic releasing hormone (TRH), Ca+2, dopamine, glucocorticoids and estradiol (Camper et al., J. Biol. Chem. (1985) 260:12246-12251). Elsholtz et al. (Science (1986) 234:1552-1557) and others have used deletion analysis of the rat prolactin promoter region to define two major regulatory regions, both of which respond to a number of these inducing/repressing molecules. Both of these regions have enhancer activity in that they function in either orientation and can activate genes driven by heterologous promoters in rats. The most proximal regulatory region is located within the first 300 bases of the 5' flanking region; the distal enhancer is approximately 1.5 kb from the transcriptional start site (Nelson et al., Nature (1986) 322:557-562).
Previously, 1 kb of 5' flanking sequence has been identified for the bovine prolactin gene. The sequence of the first 250 base pairs of the bovine prolactin 5' flanking region is highly similar to the sequence of the proximal enhancer region of the rat prolactin gene (Camper et al., DNA (1984) 3:237-249). No additional regulatory sequences analogous to the rat distal enhancer region have been reported. In fact, it has been reported that the proximal 250 base pairs contain all the sequence information necessary for regulation by inducing hormones including epidermal growth factor, thyrotropic releasing hormone, and dexamethasone (Camper et al., (supra)).
A number of groups have attempted to use prolactin regulatory sequences to effect expression of heterologous proteins. Nelson et al. (Nature (1986) 322:557) reports the coupling of the rat prolactin promoter to a heterologous gene, and the expression of the hybrid gene in tissue culture.
Palmiter and Brinster, Ann. Rev. Genetics, 20:465-499 (1986) review experiments with gene transfer into the germ-line cells of mice and with gene expression in transgenic mice. Table 4 in this reference refers to expression of a "prolactin/SV40 early region" transgene in pituitary (lactotroph) cells of transgenic mice.
Rottman et al. (PCT88/02463) discloses a method of expressing a non-prolactin gene in a transgenic animal which involves operably linking the gene to a bovine prolactin promoter and introduction into the animal. Since the prolactin promoter is relatively weak, the Rous Sarcoma Virus (RSV) enhancer element was necessary to enhance the transcription rate to an acceptable level. When the RSV enhancer was added, tissue specificity remained intact, but regulation by EGF and dexamethasone was blunted. The use of a viral enhancer is not nearly as desirable as the use of a native enhancer. However, the native bovine prolactin enhancer element was not known prior to this invention.